RNA editing facilitates the enhanced production of neoantigens during the simultaneous administration of oxaliplatin and radiotherapy in colorectal cancer

Most cases of colorectal cancers (CRCs) are microsatellite stable (MSS), which frequently demonstrate lower response rates to immune checkpoint inhibitors (ICIs). RNA editing produces neoantigens by altering amino acid sequences. In this study, RNA editing was induced artificially by chemoradiation therapy (CRT) to generate neoantigens in MSS CRCs. Altogether, 543 CRC specimens were systematically analyzed, and the expression pattern of ADAR1 was investigated. In vitro and in vivo experiments were also performed. The RNA editing enzyme ADAR1 was upregulated in microsatellite instability–high CRCs, leading to their high affinity for ICIs. Although ADAR1 expression was low in MSS CRC, CRT including oxaliplatin (OX) treatment upregulated RNA editing levels by inducing ADAR1. Immunohistochemistry analyses showed the upregulation of ADAR1 in patients with CRC treated with CAPOX (capecitabine + OX) radiation therapy relative to ADAR1 expression in patients with CRC treated only by surgery (p < 0.001). Compared with other regimens, CRT with OX effectively induced RNA editing in MSS CRC cell lines (HT29 and Caco2, p < 0.001) via the induction of type 1 interferon-triggered ADAR1 expression. CRT with OX promoted the RNA editing of cyclin I, a neoantigen candidate. Neoantigens can be artificially induced by RNA editing via an OX–CRT regimen. CRT can promote proteomic diversity via RNA editing.


ADAR1 is upregulated in CMS1 and MSI CRCs.
Whether immune-related markers are associated with any consensus molecular subtype (CMS) subgroups in CRC, a classification based on heterogeneity at the gene expression level, was assessed 9 . MSI CRCs are classified as CMS1. The relationship between the MSI status, CMS classification, and RNA editing as a potential new source of neoantigens for immunotherapy was first analyzed.
The patients with CRC classified as CMS1 showed strong immune activation, with upregulation of CD8, the surface marker of cytotoxic T cells (p < 0.01; Fig. 1A). In addition, programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) (both p < 0.001) were upregulated in CMS1 CRCs. PD-1 and PD-L1 are both known predictive markers of cancer immunotherapy, suggesting that CMS1 CRCs have high affinity to ICIs. This is primarily by virtue of which MSI CRCs are abundant in neoantigens and are classified as CMS1 10 .
Next, the expression pattern of the RNA editing enzyme ADAR1 was assessed because ADAR1 can produce neoantigens through RNA editing 8 . ADAR1 was upregulated to a larger extent in CMS1 CRCs compared with other subtypes (p < 0.01; Fig. 1B). However, amplification levels of ADAR1 did not change between subtypes, suggesting that the cause of ADAR1 upregulation in CMS1 CRCs was not genetic amplification but rather transcriptional activation. Furthermore, ADAR1 was upregulated in MSI CRCs relative to its expression in MSS CRCs (p < 0.001; Fig. 1C). Thus, ADAR1 appears to be upregulated in MSI or CMS1 CRCs, resulting in high affinity for ICIs, likely because RNA editing produces neoantigens in a posttranscriptional manner 8 . MSI CRCs possess two distinct advantages for cancer immunotherapy: high TMB based on deficient mismatch repair and epigenetic diversity based on high RNA editing activity. ADAR1 showed a positive correlation with CD8 (ρ = 0.50, p < 0.001), PD-1 (ρ = 0.51, p < 0.001), and PD-L1 (ρ = 0.61, p < 0.001; Fig. 1D), providing evidence that the upregulation of ADAR1 may be associated with the immunological response to CRC. Indeed, increased expression of ADAR1 has previously been associated with increased abundance of tumor-infiltrating lymphocytes in patients with breast cancer, which is in agreement with the present findings 11 . Patients with high RNA editing tumors treated using ICI have shown better prognosis in melanoma 6 . Altogether, these results show that artificial upregulation of ADAR1 followed by promotion of RNA editing and production of neoantigens may improve the response to ICIs in MSS CRCs. ADAR1 is upregulated in CRCs treated with CRT . Bioinformatics analysis revealed that increased expression of the RNA editing enzyme ADAR1 was associated with an increased immunogenic response. Because RNA editing can generate neoantigens, including edited CCNI 8 , the artificial upregulation of ADAR1 could contribute to improving cancer immunotherapy.
In this context, CRT was focused upon because it can generate type 1 interferon response, which promotes the expression of the RNA editing enzyme ADAR1. Indeed, both chemotherapy and radiotherapy are reported as activators of cancer immunotherapy 12,13 .
First, immunohistochemical (IHC) analysis was performed in clinical specimens. Because ADAR1 is expressed in both the nucleus and cytoplasm, ADAR1 expression levels were observed separately in each compartment. IHC analysis revealed strong staining for ADAR1 in CRCs treated with CRT (CAPOX-RT: capecitabine + OX + RT) ( Fig. 2A). ADAR1 was upregulated in the nuclei of CRT-treated CRCs compared with the normal mucosa or untreated CRCs (both p < 0.001; Fig. 2B). ADAR1 was also upregulated in the cytoplasm of CRT-treated CRCs compared with normal mucosa (p < 0.001) but not with untreated CRCs (Fig. 2C). Collectively, these results indicate that only nuclear ADAR1 was preferentially upregulated in CRT (CAPOX-RT)-treated CRCs compared with untreated CRCs. From the viewpoint of the so-called PANoptosis 14 , wherein cell death occurs while presenting cancer antigens, the location of ADAR1 is problematic. Cytoplasmic ADAR1 suppresses PANoptosis and tumor immunity consequently 15 . Despite the increase in ADAR1 expression by CRT, a noteworthy elevation is noted only in the nucleus. However, during the production of neoantigens via RNA editing  www.nature.com/scientificreports/ in the nucleus, this expression is not sufficient for the inhibition of PANoptosis. A positive correlation was also found between the expression level of ADAR1 in the nucleus and the surrounding lymphocyte population in CRT-treated CRCs (ρ = 0.74, p < 0.05, Supplementary Fig. 1). This suggests that ADAR1 expression in the nucleus has an immunogenic effect in CRT cases.
Of note, a combination therapy of OX and radiation increased the upregulation of CCNI editing to the greatest extent. OX was previously reported to be a reagent that can introduce immunogenic cell death, which supports the present results 16 .
The above-mentioned analysis was further validated using a resistant cell model (the Caco2 cells), in which the expression of ADAR1 is normally lower than that in HT29 cells. ADAR1 was upregulated in Caco2 cells treated with 5FU (p < 0.05) but not in cells treated with CPT-11 or OX (Fig. 4A). CCNI RNA editing was upregulated in Caco2 cells treated with 5FU or CPT-11 (both p < 0.05), but not in cells treated with OX (Fig. 4B). Thus, in contrast to HT29 cells, Caco2 cells showed resistance to OX in terms of its ADAR1 induction ability. ADAR1 was upregulated in Caco2 cells irradiated at a dose of 8 or 16 Gy (both p < 0.01; Fig. 4C), but CCNI RNA editing was upregulated in these cells only at a dose of 16 Gy (p < 0.05) and not at 8 Gy (Fig. 4D). These results suggest that ADAR1 induction by OX or radiation may be difficult in Caco2 because of low ADAR1 expression potential.
From a clinical viewpoint, a stabilized RNA editing induction system is generally preferred. Finally, CRT was tested as a means of stable induction of RNA editing. ADAR1 expression was promoted in Caco2 CRC cells treated with 5FU (p < 0.05), CPT-11 (p < 0.001), or OX (p < 0.001) and at a dose of 8 Gy, or with 5FU (p < 0.01), CPT-11 (p < 0.05), or OX (p < 0.001) and at a dose of 16 Gy (Fig. 4E). CCNI RNA editing was also upregulated in Caco2 CRC cells treated with OX (p < 0.05) at a dose of 8 or 16 Gy (Fig. 4F). Of note, only a combination of OX and radiation was able to upregulate CCNI editing in Caco2 cells.
CRT with OX treatment induces RNA editing more effectively than chemotherapy or radiation therapy alone. Compared with monotherapy, CRT combined with OX and radiation showed a significant upregulation of ADAR1, followed by promotion of CCNI RNA editing, in both HT29 and Caco2 cells (p < 0.05; Fig. 5A). Therefore, in vitro analyses using two CRC cell lines possessing different ADAR1 induction potential indicated that a combination of OX and radiation is the most effective for inducing RNA editing to produce neoantigens, including CCNI, as targets for immunotherapy.

CRT promotes global RNA editing in CRC cells. Whether the aforementioned trend was exclusive
to CCNI or affected other targets as well was also analyzed. Upregulation of RNA editing by chemoradiation was detected in other typical RNA editing sites in HT29 and Caco2 cells, e.g., AZIN1 (p < 0.001 and p < 0.05, respectively), GLI1 (p < 0.05 and p < 0.001, respectively), and APOBEC3D (both p < 0.01) (Fig. 5B,C). Thus, CRT-induced RNA editing appears to be a universal alteration that will promote epigenetic diversity targeted by the immune system. CAPOX-RT promotes ADAR1 expression more effectively than a FOLFOXIRI regimen. Because our cancer cell line experiments revealed that CRT induced both ADAR1 expression and RNA editing effectively relative to monotherapy, whether this phenomenon occurred in patients with CRC was also investigated using IHC analysis (Fig. 6A).
ADAR1 expression was promoted to a larger extent in CRC lesions treated with chemoradiation therapy, which included OX (CAPOX-RT), in comparison with ADAR1 expression in CRC lesions treated with a FOFOX-IRI (5FU + OX + CPT-11) chemo-regimen (p < 0.001; Fig. 6B). Analysis of clinical specimens therefore revealed that CRT, including OX (CAPOX-RT), is the best of the tested methods for accelerating RNA editing. Finally, from a clinical perspective, a possible candidate for CRT is a combination of CPT-11 and RT. We also performed CPT-11-RT or OX-RT on HT29 cells and compared their ability to induce ADAR1. OX-RT promoted expression of both p110 and p150 of ADAR1 compared to CPT11-RT (Fig. 6C). A CRT regimen including OX would be desirable for induction of ADAR1.
CRT promotes global RNA editing in a mouse model. RNA editing was accelerated by CRT in in vitro experiments; CRT induced CCNI RNA editing as neoantigens. Therefore, tests were conducted using an in vivo mouse model to confirm the induction of RNA editing by CRT (Fig. 7A). Xenografts were established using the Colon26 mouse CRC cell line on BALB/c mice; CRT (OX-RT) was performed later.
CRT could effectively inhibit tumor growth in the mouse model (p < 0.001, Fig. 7B). It also induced the upregulation of CD8 and PD-L1 expression (both p < 0.01) in xenograft tumors, which resulted in high affinity to ICIs (Fig. 7C). ADAR1 was effectively upregulated (p < 0.05) in the CRT group, followed by AZIN1 (p < 0.001) and CCNI (p < 0.05) editing (Fig. 7D). Thus, RNA editing was effectively induced in an immune-proficient mouse model using CRT. Moreover, treatment with CRT, which included OX, effectively produced accelerated immunoreactivity and neoantigens in combination.
CRT induces RNA editing and produces edited proteins as neoantigens. The mechanism by which RNA editing is induced by CRT was elucidated. CRC cells are known to produce type 1 interferon (IFN) 17 . In this study, IFNα (p < 0.01) and IFNβ (p < 0.001) were induced by an OX-RT CRT regimen (Fig. 8A). ADAR1 is already known to be induced by type 1 IFN 18 ; type 1 IFN likely activates ADAR1 expression and RNA editing by CRT via the autocrine system (Fig. 8B).
Overall, this study presented new evidence that showed that CRT can generate neoantigens and promote proteomic diversity via RNA editing (Fig. 8C). This technology may facilitate cancer immunotherapy, particularly in MSS CRC, which was previously excluded from the induction of ICIs.    www.nature.com/scientificreports/ To the best of our knowledge, this is the first report to demonstrate that CRT can induce RNA editing and upregulate epigenetic diversity. Previous studies have demonstrated that chemotherapy or radiation shows high affinity to ICIs 12,13 ; however, the mechanism by which these effects are orchestrated has yet to be determined. The upregulation of RNA editing followed by production of neoantigens was speculated to be the basis of this phenomenon in CRC cell lines. CCNI RNA editing induced by CRT may therefore represent a new method by which epigenetic diversity can be controlled for the purpose of cancer immunotherapy. AZIN1, GLI1, and APOB-EC3D RNA editing, which are the typical editing targets of ADAR1, were upregulated depending on ADAR1 expression by CRT. IHC analyses also showed that ADAR1 was upregulated in patients with rectal cancer treated with CAPOX-RT. Indeed, CRT tended to upregulate RNA editing, suggesting that neoantigens are generated for cancer immunotherapy via CRT.
Particularly for locally advanced rectal cancer, the combination of CRT and immunotherapy may be an improvement over the "watch and wait" strategy. In clinical trials (VOLTAGE-A), the combination of preoperative CRT (Cape-RT) and ICI for the treatment of locally advanced rectal cancer has improved the pathological complete response rate 25,26 . This may have been caused, at least partially, by epigenetic induction of neoantigens www.nature.com/scientificreports/ via RNA editing. Our data suggest that the addition of OX to a Cape-RT regimen, i.e., CAPOX-RT, produces more neoantigens and thereby improves the response to ICI. Moreover, OX has already been reported as a drug that can induce immunogenic cell death 16 . Simultaneous, rather than sequential, administration of OX, RT, and ICI is required to change cold CRC to hot CRC. CRT induces inflammatory interferons, but despite the increase in ADAR1 expression, it is only considerably elevated in the nucleus; therefore, the inflammatory cell death induced by the enhanced protocol is expected to promote the neo-antigen specific immune response by converting tumors from cold to hot. OX-RT did not interfere with ZBP1, RIP1, and RIP3, accelerators of PANoptosis, which is in agreement with our theory (Supplementary Fig. 3).
A previous study reported that loss of ADAR1 in tumors enhances the response to ICIs and overcomes resistance to immunotherapy; however, this study did not examine the correlation between actual RNA editing level changes and immunity 27 . In a recent report, a melanoma group with high RNA editing levels showed a better response rate to ICI 6 . Because immunoreactions to cancer cells differ from innate immunity, it will also be important to use RNA sequencing and further explore neoantigens to establish approaches using precision medicine. Indeed, a limitation of our study was that whether immune cells were responsible for targeting the CCNI edited by CRT (as it was in melanoma) could not be confirmed 6 . Edited CCNI is presented on HLA-A*02 8 ; thus, it is difficult to investigate this phenomenon in a mouse model. Our future studies will focus on the treatment of CRC with CAPOX-RT + ICI using a humanoid mouse.
In summary, this study provides novel evidence of a method to control epigenetic diversity using CRTmeditated RNA editing. This study highlights the biological and clinical significance of RNA editing for immunotherapy in patients with CRC, especially in locally advanced rectal cancer. Our findings suggest that CRT with www.nature.com/scientificreports/ OX is an effective treatment for accelerating immunoreactivity for ICIs. In particular, this technology may be useful as a watch and wait therapy in patients with rectal cancer.

Materials and methods
Patients and sample collection. This study included the analysis of 543 CRC cases, which comprised 512 CRC specimens from the Cancer Genome Atlas (TCGA) dataset (https:// cance rgeno me. nih. gov/) 28 RNA editing site-specific quantitative polymerase chain reaction (RESSq-PCR). The degree of RNA editing of AZIN1, GLI1, and APOBEC3D was analyzed using RESSq-PCR as previously reported 33 . In brief, specific primers for the wild-type and edited AZIN1, GLI1, and APOBEC3D sequences were designed. Based on the difference in the Ct values, the ratios between the edited and wild-type sequences were calculated using the formula 2 −(Ct Edited − Ct Wild-type) . Primer sequences for the PCRs are shown in Supplementary Cell lines. The HT29 and Caco2 CRC cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Tokyo, Japan) 3 months before the experiment began. These cells were authenticated annually by the JCRB Cell Bank using short tandem repeat analysis. All cell lines were cultured according to the manufacturer's specifications. All experiments were performed using cells that did not exceed 15-20 passages.
Chemotherapy and radiation therapy. Cells were cultured for 48 h in a medium to which 5 μM of 5-FU, 5 μM of CPT-11, or 30 μM of OX was added. Radiation therapy was administered at a single dose of 8 or 16 Gy.

Radar chart.
A radar chart was used to visualize ADAR1 expression and RNA editing levels for multiple comparisons. The levels of ADAR1 expression and CCNI editing, respectively, were compared among the chemotherapy, radiation, and chemoradiation (OX + radiation) groups.

Statistical analysis.
Results are shown as means ± standard deviation. JMP software (ver. 10.0, SAS Institute Inc., Cary, NC, USA) was used to perform statistical analyses. Differences between groups were estimated using the Wilcoxon signed rank test, χ 2 test, and Steel test, as appropriate. Correlations between groups were analyzed using Spearman's rank correlation analysis. All p values were two sided and those lesser than 0.05 were considered statistically significant.
Ethics approval and consent to participate. Written informed consent was obtained from each patient, and the institutional review board (The Ethics Committee of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital) approved the study .